Four Individually Identified Paired Dopamine Neurons Signal Taste Punishment in Larval Drosophila

Reviewer #1 (Public Review):

Summary:
In this paper, Weber et al. investigate the role of 4 dopaminergic neurons of the Drosophila larva in mediating the association between an aversive high-salt stimulus and a neutral odor. The 4 DANs belong to the DL1 cluster and innervate non-overlapping compartments of the mushroom body, distinct from those involved in appetitive associative learning. Using specific driver lines, they show that activation of the DAN-g1 is sufficient to mimic an aversive memory and it is also necessary to form a high-salt memory of full strength, although optogenetic silencing of this neuron only partially affects the performance index. The authors use calcium imaging to show that the DAN-g1 is not the only one that responds to salt. DAN-c1 and d1 also respond to salt, but they seem to play no role in the assays tested. DAN-f1, which does not respond to salt, is able to lead to the formation of memory (if optogenetically activated), but it is not necessary for the salt-odor memory formation in normal conditions. However, silencing of DAN-f1 together with DAN-g1, enhances the memory deficit of DAN-g1.

Strengths:
The paper therefore reveals that also in the Drosophila larva as in the adult, rewards and punishments are processed by exclusive sets of DANs and that a complex interaction between a subset of DANs mediates salt-odor association.
Overall, the manuscript contributes valuable results that are useful for understanding the organization and function of the dopaminergic system. The behavioral role of the specific DANs is accessed using specific driver lines which allow for testing of their function individually and in pairs. Moreover, the authors perform calcium imaging to test whether DANs are activated by salt, a prerequisite for inducing a negative association with it. Proper genetic controls are carried across the manuscript.

Weaknesses:
The authors use two different approaches to silence dopaminergic neurons: optogenetics and induction of apoptosis. The results are not always consistent, and the authors could improve the presentation and interpretation of the data. Specifically, optogenetics seems a better approach than apoptosis, which can affect the overall development of the system, but apoptosis experiments are used to set the grounds of the paper.

The physiological data would suggest the role of a certain subset of DANs in salt-odor association, but a different partially overlapping set seems to be necessary. This should be better discussed and integrated into the author's conclusion. The EM data analysis reveals a non-trivial organization of sensory inputs into DANs and it is hard to extrapolate a link to the functional data presented in the paper.

Reviewer #2 (Public Review):

Summary:
In this work, the authors show that dopaminergic neurons (DANs) from the DL1 cluster in Drosophila larvae are required for the formation of aversive memories. DL1 DANs complement pPAM cluster neurons which are required for the formation of attractive memories. This shows the compartmentalized network organization of how an insect learning center (the mushroom body) encodes memory by integrating olfactory stimuli with aversive or attractive teaching signals. Interestingly, the authors found that the 4 main dopaminergic DL1 neurons act redundantly, and that single-cell ablation did not result in aversive memory defects. However, ablation or silencing of a specific DL1 subset (DAN-f1,g1) resulted in reduced salt aversion learning, which was specific to salt but no other aversive teaching stimuli were tested. Importantly, activation of these DANs using an optogenetic approach was also sufficient to induce aversive learning in the presence of high salt. Together with the functional imaging of salt and fructose responses of the individual DANs and the implemented connectome analysis of sensory (and other) inputs to DL1/pPAM DANs, this represents a very comprehensive study linking the structural, functional, and behavioral role of DL1 DANs. This provides fundamental insight into the function of a simple yet efficiently organized learning center which displays highly conserved features of integrating teaching signals with other sensory cues via dopaminergic signaling.

Strengths:
This is a very careful, precise, and meticulous study identifying the main larval DANs involved in aversive learning using high salt as a teaching signal. This is highly interesting because it allows us to define the cellular substrates and pathways of aversive learning down to the single-cell level in a system without much redundancy. It therefore sets the basis to conduct even more sophisticated experiments and together with the neat connectome analysis opens the possibility of unraveling different sensory processing pathways within the DL1 cluster and integration with the higher-order circuit elements (Kenyon cells and MBONs). The authors' claims are well substantiated by the data and clearly discussed in the appropriate context. The authors also implement neat pathway analyses using the larval connectome data to its full advantage, thus providing network pathways that contribute towards explaining the obtained results.

Weaknesses:
While there is certainly room for further analysis in the future, the study is very complete as it stands. Suggestions for clarification are minor in nature.

Reviewer #3 (Public Review):

The study of Weber et al. provides a thorough investigation of the roles of four individual dopamine neurons for aversive associative learning in the Drosophila larva. They focus on the neurons of the DL-1 cluster which already have been shown to signal aversive teaching signals. However, the authors go far beyond the previous publications and test whether each of these dopamine neurons responds to salt or sugar, is necessary for learning about salt, bitter, or sugar, and is sufficient to induce a memory when optogenetically activated. In addition, previously published connectomic data is used to analyze the synaptic input to each of these dopamine neurons. The authors conclude that the aversive teaching signal induced by salt is distributed across the four DL-1 dopamine neurons, with two of them, DAN-f1 and DAN-g1, being particularly important. Overall, the experiments are well designed and performed, support the authors' conclusions, and deepen our understanding of the dopaminergic punishment system.

Strengths:
1. This study provides, at least to my knowledge, the first in vivo imaging of larval dopamine neurons in response to tastants. Although the selection of tastants is limited, the results close an important gap in our understanding of the function of these neurons.

2. The authors performed a large number of experiments to probe for the necessity of each individual dopamine neuron, as well as combinations of neurons, for associative learning. This includes two different training regimens (1 or 3 trials), three different tastants (salt, quinine, and fructose) and two different effectors, one ablating the neuron, the other one acutely silencing it. This thorough work is highly commendable, and the results prove that it was worth it. The authors find that only one neuron, DAN-g1, is partially necessary for salt learning when acutely silenced, whereas a combination of two neurons, DAN-f1 and DAN-g1, are necessary for salt learning when either being ablated or silenced.

3. In addition, the authors probe whether any of the DL-1 neurons is sufficient for inducing an aversive memory. They found this to be the case for three of the neurons, largely confirming previous results obtained by a different learning paradigm, parameters, and effector.

4. This study also takes into account connectomic data to analyze the sensory input that each of the dopamine neurons receives. This analysis provides a welcome addition to previous studies and helps to gain a more complete understanding. The authors find large differences in inputs that each neuron receives, and little overlap in input that the dopamine neurons of the "aversive" DL-1 cluster and the "appetitive" pPAM cluster seem to receive.

5. Finally, the authors try to link all the gathered information in order to describe an updated working model of how aversive teaching signals are carried by dopamine neurons to the larva's memory center. This includes important comparisons both between two different aversive stimuli (salt and nociception) and between the larval and adult stages.

Weaknesses:
1. The authors repeatedly claim that they found/proved salt-specific memories. I think this is problematic to some extent.

1a. With respect to the necessity of the DL-1 neurons for aversive memories, the authors' notion of salt-specificity relies on a significant reduction in salt memory after ablating DAN-f1 and g1, and the lack of such a reduction in quinine memory. However, Fig. 5K shows a quite suspicious trend of an impaired quinine memory which might have been significant with a higher sample size. I therefore think it is not fully clear yet whether DAN-f1 and DAN-g1 are really specifically necessary for salt learning, and the conclusions should be phrased carefully.

1b. With respect to the results of the optogenetic activation of DL-1 neurons, the authors conclude that specific salt memories were established because the aversive memories were observed in the presence of salt. However, this does not prove that the established memory is specific to salt - it could be an unspecific aversive memory that potentially could be observed in the presence of any other aversive stimuli. In the case of DAN-f1, the authors show that the neuron does not even get activated by salt, but is inhibited by sugar. Why should activation of such a neuron establish a specific salt memory? At the current state, the authors clearly showed that optogenetic activation of the neurons does induce aversive memories - the "content" of those memories, however, remains unknown.

2. In many figures (e.g. figures 4, 5, 6, supplementary figures S2, S3, S5), the same behavioural data of the effector control is plotted in several sub-figures. Were these experiments done in parallel? If not, the data should not be presented together with results not gathered in parallel. If yes, this should be clearly stated in the figure legends.